The striatum plays a key role in motor activity/coordination and goal-directed, habitual learning. Normal striatal drive of motor activity requires precisely balanced opposing outputs from two types of striatal medium spiny neurons (MSNs) that express D1- and D2-dopamine(DA) receptors. A complex signaling cross-talk between glutamate and DA in D1- and D2-MSNs involves on demand Ca2+-dependent endocannabinoid (eCB) synthesis. Thus, DA, glutamate and eCBs collaborate to balance short- and long-term control of the two striatal output pathways by engaging distinct signaling mechanisms in the two MSN subtypes. Disruption of these mechanisms can induce motor deficits (e.g., Parkinson's Disease) or other abnormal striatal-based behaviors. Ca2+/calmodulin-dependent protein kinase II (CaMKII) has diverse bidirectional roles controlling excitatory synapses in hippocampus, cortex and cerebellum. While CaMKII is expressed in both striatal MSN subtypes, suggesting that it regulates excitatory inputs to striatal MSNs and motor activity, the precise functions of striatal CaMKII are poorly understood. Our analyses of knockin mutant mice with the Thr286 autophosphorylation site in CaMKII? replaced by Ala (T286A-KI mice) revealed specific roles for CaMKII in long- and short term eCB- dependent control of excitatory inputs to D1- and D2-MSNs. We also found that CaMKII? associates with and phosphorylates diacylglycerol lipase ? (DGL?), the rate-limiting enzyme for Ca2+-dependent synthesis of the most abundant brain eCB, 2-arachidonyl glycerol (2-AG). In addition, baseline hyperactivity of T286A-KI mice can be rescued by inhibiting 2-AG breakdown. These initial findings strongly support a novel hypothesis that CaMKII is a critical link between postsynaptic Ca2+ and the initiation of 2-AG signaling that controls striatal synapses and striatal based-behaviors. We also created novel transgenic eAC3I mice that selectively express a short CaMKII inhibitor peptide fused to eGFP in striatal MSNs. Three specific aims will exploit unique features of T286A-KI and eAC3I mice to test specific hypotheses about the roles of striatal CaMKII autophosphorylation and activity. 1. Test the hypothesis that Ca2+-dependent 2-AG synthesis is modulated by CaMKII. We will identify sites of phosphorylation and CaMKII-binding domains in DGL?. DGL? phosphorylation, DGL? activity and 2-AG synthesis will be investigated in heterologous cells and in striatal slices from WT, T286A-KI and eAC3I mice. 2. Test the hypothesis that CaMKII modulates eCB-dependent synaptic regulation in striatal MSNs. Short and long-term roles of CaMKII will be determined by comparing the properties of excitatory synaptic inputs to D1- and D2-MSNs in striatal slices from WT, T286A-KI and eAC3I mice, and by using CaMKII inhibitor peptides. 3. Test the hypothesis that CaMKII modulates eCB-dependent motor activity. We will evaluate motor activity and coordination under basal conditions and following pharmacological modulation of 2-AG metabolism in WT, T286A-KI and eAC3I mice. PUBLIC HEALTH RELEVANCE: Normal movement and coordination is dependent on a brain region called the striatum, and damage to the striatum gives rise to movement disorders such as Parkinson's Disease, Huntington's Disease and several forms of ataxia. The principle neuronal cells in striatum (medium spiny neurons) integrate input signals from motor cortex and thalamus to provide precisely balanced output to other brain regions that control motor activity. This project investigates molecular mechanisms that control the strength of these input signals, with the goal of identifying novel therapeutic targets to treat movement disorders.